Abstract Klebsiella pneumoniae (Kpn) is a leading cause of Gram-negative nosocomial infections and is associated with a high mortality rate. Antibiotic resistance is a growing issue among the Enterobacteriaceae and of the Enterobacteriaceae Kpn is the most prevalent ESBL (extended spectrum ?-lactamase) and CRE (carbapenem resistant Enterobacteriaceae) isolate. The increasing prevalence of antibiotic-resistant Kpn only serves to compound its clinical importance and to complicate treatment options. Kpn strains are broadly classified as hypervirulent (hv) or classical, with hv strains typically causing community acquired liver abscess infections and invasive infections. Classical strains are more typically associated with nosocomial infections. The hv strains have a hypermucoviscous (Hmv+) phenotype due in part to hyper-production of capsule. Recent genomic analyses of large strain sets have advanced our understanding of the different subgroups of Kpn, yet we still do not have a clear picture of the genetic traits required by each group to cause disease. Despite significant diversity in the capsule type and amount of capsule, capsule is an essential virulence factor for all Kpn. Capsule production in Kpn is tightly regulated and a number of transcriptional regulators have been identified, including RcsB and RmpA. Using an Hmv+ strain (KPPR1) our lab recently identified three new transcription factors (RmpC, KvrA and KvrB) required for full expression of capsule synthesis genes and for full virulence. All five regulators cause similar decreases in expression of one of the capsule promoters. In murine infections, these mutants each are attenuated, but curiously not to the same degree. In addition, some of the regulatory mutants retain the Hmv+ phenotype whereas others are Hmv- suggesting they play roles beyond controlling expression of the cps locus. We recently extended these findings to an hv strain with a different capsule type and will also test findings in an ST258 strain. Our data suggest that some of the findings from KPPR1 will be conserved between strains of Kpn. Thus, we hypothesize these regulators regulate multiple and varying steps in the pathway producing capsule, Hmv, and possibly additional virulence determinants. To address this hypothesis we propose the following aims: Aim 1. Regulation of capsule production. RcsB, RmpA, RmpC, KvrA and KvrB all affect production of capsule yet little is known of the mechanism of this regulation. We will address this by examining regulatory effects at the known cps promoters, determining if the regulators function independently or dependently, and which directly bind the cps promoter. Aim 2. Determine the regulons of each transcriptional regulator. As each of the five regulators affects capsule production and yet the virulence phenotypes differ, they likely regulate additional virulence determinants. Thus, we will use RNA-seq to identify genes impacted by each of these transcriptional regulators. Follow-up studies will include in vivo and in vitro virulence studies to better understand the contribution of each regulator to pathogenesis. Collectively, we aim to correlate pathogenesis with the genes affected by the regulators. ! !